Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance

Abstract As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant‐specific Ca2+ sensors, calmodulin and calmodulin‐like (CML) proteins, function as members of the second‐messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two‐hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+‐dependent way in a knockdown study. Detailed investigations indicated that several defence‐related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45‐ and GbCML50‐modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt‐tolerant cotton cultivars by genetic engineering and molecular breeding.


| INTRODUC TI ON
Calcium (Ca 2+ ) works as a second messenger participating in signal transduction to regulate plant growth, development and multienvironmental responses through regulation of various cellular processes (Berridge et al., 2000;Sanders et al., 1999).The variation of cellular Ca 2+ levels in plants shows spatiotemporal properties according to various environmental stimuli and stress conditions (Aldon et al., 2018;Bender & Snedden, 2013).In plants, the rapid movement of Ca 2+ into the cytosol is a key event activated by the perception of pathogen-associated molecular patterns (PAMPs) such as during the early stage of pathogen attack, or by perception of abiotic stresses such as drought or exposure to ozone (Köster et al., 2022;Kudla et al., 2018;Lecourieux et al., 2006).To perceive a stimulus, the Ca 2+ messenger is decoded and relayed by Ca 2+ -binding proteins (also named Ca 2+ sensors) to activate cellular responses (Steinhorst & Kudla, 2014).Four groups of Ca 2+ sensors have been identified in plants: calmodulin (CaM), calmodulin-like (CML), calcineurin B-like (CBL) and the Ca 2+ -dependent protein kinase (CDPK/CPK) proteins (Batistič & Kudla, 2012;McCormack et al., 2005;Zhu et al., 2015).
Many Ca 2+ sensors possess a highly conserved helix-loop-helix motif (also named EF-hand) that contains 29 amino acids and forms a loop structure; the 12 central residues can bind one Ca 2+ ion (La Verde et al., 2018).The expression patterns of CMLs are regulated by environmental stimuli or various plant developmental stages, indicating that CMLs may play specific roles in growth, development and different stress responses in plant (Cheval et al., 2013;Ranty et al., 2016;Zhu et al., 2015).
CaMs/CMLs are involved in plant immunity by activating CaM/ CML-binding transcription factors (TFs) and regulating plant defence in several plant species (Du et al., 2009;Galon et al., 2008;Wang et al., 2009Wang et al., , 2011)).CaMs/CMLs also target a large group of downstream CaM-binding proteins (CBPs), including transporters, protein phosphatases, protein kinases and metabolic enzymes, and form complexes in Ca 2+ -dependent or -independent ways (Reddy et al., 2011;Zeng et al., 2015Zeng et al., , 2023)).Several members of CMLs in Arabidopsis thaliana, such as CML8, CML9, CML41 and CML43, have been shown to positively regulate plant resistance to Pseudomonas syringae pv.tomato (Pst) DC3000 (Chiasson et al., 2005;Leba et al., 2012;Xu et al., 2017;Zhu et al., 2017).CML46 and CML47 also modulate salicylic acid (SA) accumulation and enhance Arabidopsis resistance to the pathogen P. syringae pv.maculicola (Pma) (Lu et al., 2018).CML orthologues positively regulating plant disease resistance have also been discovered in other plant species.For example, overexpression of soybean (Glycine max) SCaM4 and SCaM5 genes in Arabidopsis promotes the resistance of transgenic plants to P. syringae and Phytophthora sojae (Heo et al., 1999;Park et al., 2004).Silencing the pathogen-induced NtCaM13, an orthologue of Arabidopsis CaM4 and CaM5, in Nicotiana benthamiana results in elevated susceptibility of plants to necrotrophic pathogens such as Alternaria tenuissima and Phomopsis longicolla (Takabatake et al., 2007).In addition, wheat (Triticum aestivum) TaCML36 and pepper (Capsicum annuum) CaCML13 also enhance immune responses to Rhizoctonia cerealis (Lu et al., 2019) and Ralstonia solanacearum in plants (Shen et al., 2020), respectively.However, tomato (Solanum lycopersicum) SlCML55 decreased defence against Phytophthora pathogens by inhibiting the SA signalling pathway (Zhang, Zou, et al., 2022).These studies suggest that different CaM and CML genes may function as positive or negative regulators of pathogen resistance in different plant species.
Other phytohormones, including jasmonic acid (JA), SA and strigolactones (SLs), also participate in plant resistance to Verticillium infection | 3 of 17 YI et al. (Jia et al., 2022;Song et al., 2020;Yi et al., 2023).JA enhances plant resistance to V. dahliae infection by stimulating the downstream myelocytomatosis 2 (MYC2) and plant defence factor 1.2 (PDF1.2) gene expression (He et al., 2018).The SA-regulated defence pathway displays both cooperative and inhibitory effects on JA signalling pathway (Wang et al., 2020).The non-expressor of pathogenesis-related protein 1 (NPR1) and NPR1-like protein 3 (NPR3)/NPR4 receptors perceive the SA-mediated responses to prompt the expression of pathogenesis-related (PR) genes, such as PR1 and PR5, which in turn increases plant resistance against V. dahliae infection in some plant species, including cotton (Liu et al., 2020;Wu et al., 2012;Yan et al., 2016).On the other hand, the suppression of GhWRKY70A05a, a negative regulator of cotton resistance to V. dahliae, improved cotton resistance to V. dahliae infection by inhibiting the SA signalling pathway and promoting the JA signalling pathway (Xiong et al., 2019).
Recently, some Ca 2+ -dependent proteins were reported to be associated with cotton Verticillium wilt resistance.For example, the knockdown of GhCPK33 led to decreased resistance to V. dahliae infection by the down-regulation of JA biosynthesis in cotton (Hu et al., 2018), while the myeloblastosis 108 (MYB108) TF forms a positive feedback loop to promote the transcription of GhCML11 in a Ca 2+ -dependent way in enhancing V. dahliae resistance (Cheng et al., 2016).Mutation in plant-specific master TF-encoding CBP60g or systemic acquired resistance-deficient 1 (SARD1), which modulate many defence-related genes in immunity and are targeted by a V. dahliae secretory protein 41 (VdSCP41), caused compromised resistance of Arabidopsis mutants against V. dahliae (Qin et al., 2018).Moreover, silencing of GhCBP60b, also a target of VdSCP41, compromised resistance to V. dahliae in cotton (Qin et al., 2018).Acetylation of GhCaM7 enhances resistance to V. dahliae through increasing its interaction with the osmotin protein GhOSM34 in cotton, which then induces multiple disease resistance signalling pathways (Zhang et al., 2023).
However, the mechanisms Ca 2+ /CaM/CML-regulated pathways underlying Verticillium wilt resistance remain unknown in cotton.
In this study, by analysing root transcriptome data, we identified a V. dahliae-responsive gene, GbCML45, from roots of the island cotton (Gossypium barbadense) and found that it positively regulated Verticillium wilt resistance in both cotton and Arabidopsis.We found that GbCML45 can interact with its close homologue GbCML50.Further investigations revealed that GbCML50 also has positive regulatory role in Verticillium wilt resistance in cotton, where both GbCML45 and GbCML50 genes modulate the defence-related SA and ET signalling pathways, ROS burst, lignin synthesis and callose deposition to enhance cotton resistance to V. dahliae infection.

| V. dahliae infection induces the expression of GbCML45 in cotton and knockdown of GbCML45 decreases cotton resistance to Verticillium wilt
To screen for candidate genes related to Verticillium wilt disease resistance in cotton, we analysed the root transcriptome data of two chromosome segment substitution 1 (CSSL1, resistant cultivar) and CSSL4 (susceptible cultivar) lines after being inoculated with V. dahliae (Zhang, Liu, et al., 2022).We found that the fragments per kilobase of transcript per million mapped reads (FPKM) values of the Gbscaffold26027.2.0 gene (named GbCML45 hereafter) were increased by about 10-and 30-fold in the roots of resistant CSSL1 and susceptible CSSL4 cultivars, respectively, after their inoculation with V. dahliae for about 60 days in the disease nursery (Figure 1a,b).
The phylogenetic tree analysis showed that GbCML45 has a conserved evolutionary relationship with those from other plant species that contain the EF-hand domain (Figure S1a,b).Additionally, Gb/GhCML45 was highly expressed in cotton roots, but expressed at low levels in stems and leaves in both the island (resistant cultivar Hai7124) and upland (Gossypium hirsutum, susceptible cultivar Jimian11) cotton plants (Figures S1c and S2a).
To confirm the up-regulation of CML45 expression after V. dahliae infection in our experimental system, we conducted a reverse transcription-quantitative PCR (RT-qPCR) assay to detect the expression levels of GbCML45 and GhCML45 in roots of the island and upland cotton plants in a time course after infection by a V. dahliae spore suspension (10 7 spores/mL).There was an increase by approximately five-and three-fold in the expression levels of CML45 in the roots of both Hai7124 and Jimian11 plants at 0.25 and 3 days post-inoculation (dpi), respectively (Figures 1c   and S2b).To assess the role of CML45 in V. dahliae resistance, we knocked down the expression of CML45 in both Hai7124 and Jimian11 by using the virus-induced gene silencing (VIGS) method in cotton.The expression levels of GbCML45 and GhCML45 were significantly lower in TRV:GbCML45 and TRV:GhCML45 plants than in the control TRV:00 plants (Figures 1d and S2c).A marker gene, chloroplastos alterados (CLA), was silenced (TRV:CLA) to monitor the efficiency of VIGS at the same time, which indicated the proper action of the VIGS system through the albino phenotype of the newly grown true leaves (Figures 1e and S2d) (Gao et al., 2017).After their infection with V. dahliae for 21 days, there were more severe Verticillium wilt disease symptoms for both the TRV:GbCML45 and TRV:GhCML45 cotton plants (Figures 1f and   S2e) and higher disease index values (Figures 1g and S2f) than TRV:00 plants.Additionally, the TRV:GbCML45 and TRV:GhCML45 plants had more brown spots and lesion areas than TRV:00 plants in their stem vascular tissues and more biomass of V. dahliae in stems (Figures 1h,i and S2g,h).These results indicated that the V. dahliae-induced GbCML45/GhCML45 positively regulated cotton resistance to V. dahliae infection.

| GbCML45 interacts with GbCML50 and some TFs, and forms a homodimer
To identify some potential GbCML45-interacting proteins in cotton, we constructed a yeast cDNA library using RNA from V. dahliaeinfected Hai7124 cotton roots and used GbCML45 as bait to perform a yeast two-hybrid (Y2H) assay.Multiple putative GbCML45interactors were identified, and 56 positive yeast colonies were selected for sequencing and annotation.Three GbCML45-interacting proteins were selected for further study: CML50, ERF and related to apetala (AP) 2.3 (RAP2.3)TFs (Table S1).Yeast colonies containing activation domain (AD)-GbCML45 plus binding domain (BD)-GbCML50, BD-GbAP2-ERF or BD-GbRAP2.3were able to grow in the presence of 3-aminotriazole (3AT) and X-Gal, verifying the interaction between GbCML45 and these identified proteins (Figure 2a).A bimolecular fluorescence complementation (BiFC) assay was conducted to further confirm the interaction of GbCML45 with GbCML50, GbAP2-ERF and GbRAP2.3 in vivo.Yellow fluorescence was found in the cell membrane and nuclei when GbCML45-cYFP and GbCML50-nYFP were co-expressed in Nicotiana benthamiana epidermal cells, while fluorescence was found only in the cell membrane when GbCML45-cYFP was co-expressed with GbAP2-ERF-nYFP or GbRAP2.3-nYFP,strengthening the interaction of GbCML45 with GbCML50, GbAP2-ERF and GbRAP2.3 in vivo (Figure 2b).
In addition, we found that GbCML45 could interact with itself and form a homodimer in the Y2H assay, while such homodimer formation was not detected in the case of GbCML50 in our Y2H assay (Figure S3a).These results were also verified by the BiFC assay where the homodimer formation of GbCML45 was observed as indicated by the yellow fluorescence in epidermal cells when GbCML45-cYFP and GbCML45-nYFP were co-expressed.However, no fluorescence was detected in epidermal cells having GbCML50-cYFP and GbCML50-nYFP co-expressed (Figure S3b).

| Knockdown of GbCML50 decreases Verticillium wilt resistance in cotton
Because GbCML50 interacts with GbCML45, we asked if GbCML50 is also involved in regulating the cotton response to V. dahliae infection.
We knocked down GbCML50 in the resistant Hai7124 cultivar using the VIGS method and assessed the responses of silenced plants to V. dahliae infection (Figure 3a,b).We found that TRV:GbCML50 plants were more damaged by Verticillium wilt than TRV:00 plants through observation of disease symptom and disease index (Figure 3c,d).Additionally, the TRV:GbCML50 plants displayed deeper brown spots and more biomass of V. dahliae than TRV:00 plants (Figure 3e,f).These results demonstrated that the knockdown of GbCML50 increased cotton susceptibility to V. dahliae infection, and thus that GbCML50 has a positive role in cotton Verticillium wilt resistance similar to its interacting partner GbCML45.
We also knocked down the GbAP2-ERF and GbRAP2.3 in similar manner to examine whether they function in cotton Verticillium wilt resistance (Figure 3g,h).After infection with V. dahliae, TRV:GbAP2-ERF and TRV:GbRAP2 plants exhibited more severe disease symptoms than TRV:00 control plants; however, no significant differences in disease index value were recorded when comparing TRV:GbAP2-ERF and TRV:GbRAP2 plants with TRV:00 plants (Figure 3i,j).Furthermore, the TRV:GbAP2-ERF and TRV:GbRAP2 cotton plants showed no obvious differences in terms of brown spots and biomass of V. dahliae in stems as compared with TRV:00 plants (Figure 3k,l).On the basis of these findings, we subsequently focused on the mechanisms of GbCML45 and GbCML50 in regulating cotton resistance to Verticillium wilt.

| Subcellular localization analysis and transcriptional activation assays of GbCML45 and 50
The subcellular localization of GbCML45 and GbCML50 in plant cells was investigated using an infiltration assay of the GFP-fused constructs in N. benthamiana leaves.Green fluorescence detected in both cytoplasm and nuclei of epidermal cells supported the expression of the GbCML45-GFP and GbCML50-GFP constructs in these organelles (Figure 4).

| Both GbCML45 and GbCML50 positively regulate Verticillium wilt resistance in Arabidopsis as their Arabidopsis homologues AtCML46 and AtCML49
To examine the functions of GbCML45 and GbCML50 in regulating plant resistance to Verticillium wilt, we ectopically expressed each in Arabidopsis wild-type (WT) plants (Figure S4a).Transgenic plants ectopically expressing GbCML45 or GbCML50 showed less severe disease symptoms, and lower disease index values and V. dahliae biomass than WT plants (Figure S4b-f).These results demonstrated the positive roles of GbCML45 and GbCML50 genes in regulating plant resistance to V. dahliae infection in the heterologous Arabidopsis system as well.
We also identified the closest orthologues of GbCML45 and GbCML50 in Arabidopsis, namely AtCML46 (AT5G39670) and AtCML49 (AT3G10300), and their corresponding loss-of-function mutants.The Atcml46 and Atcml49 mutants were then challenged with V. dahliae infection.At 14 dpi, more severe disease symptoms and higher disease index values were recorded for the Atcml46 and Atcml49 mutant plants than WT plants (Figure S4g,h).The Atcml46 and Atcml49 plants had more biomass of V. dahliae than WT plants (Figure S4i).These data suggest that GbCML45 and GbCML50 from cotton and their homologues from Arabidopsis have a conserved function, at least in terms of plant resistance to V. dahliae infection.

| V. dahliae infection induces GbCML45-and GbCML50-mediated Ca 2+ influx
CMLs function as Ca 2+ sensors in the earliest cellular responses to many abiotic/biotic stresses in plants (Zeng et al., 2023)

| GbCML45 and GbCML50 enhance cotton Verticillium wilt resistance dependent on SA and ET signalling pathways
Hormones such as SA, ET and methyl JA (MeJA) are associated with plant defence responses (Gupta et al., 2020;Li et al., 2019), and their crosstalk plays critical roles in Ca 2+ -mediated immunity in plant (Heyer et al., 2022;Köster et al., 2022).Thus, we checked the expres-

| GbCML45 and GbCML50 enhance Verticillium wilt resistance by inducing ROS and accumulation of NO, lignin and callose
Increased intracellular Ca 2+ and extensive interplay between Ca 2+ -CaMs and ROS are common early cellular events to amplify plant immune signalling pathways in response to pathogen infection or elicitor treatment (Marcec et al., 2019;Zeng et al., 2023).To test the role of ROS in GbCML45/50-mediated Verticillium wilt resistance, a 3,3′-diaminobenzidine (DAB) staining assay was performed.the expression levels of ROS production-related genes GbRbohD, GbRboh5 and nucleoredoxin (GbNRX1) were also reduced in the TRV:GbCML45 and TRV:GbCML50 cotton roots after 48 hpi with V. dahliae (Figure 7c).Similarly, the expression levels of NO synthesisrelated gene NO associated 1 (GbNOA1) and the contents of NO were more reduced in TRV:GbCML45 and TRV:GbCML50 plants than in TRV:00 plants (Figure 7c).These results indicated that GbCML45 and GbCML50 play an important role in regulating ROS and NO levels in plants cells to modulate cotton Verticillium wilt resistance at the early stage of V. dahliae infection.
Deposition of lignin and callose in the cell wall is important for plant defence against pathogen invasion as these components form a physical barrier to prevent the diffusion of pathogen-produced toxins (Guo et al., 2016;Huang et al., 2021;Mbiza et al., 2022;Zhang et al., 2019).
We asked if GbCML45 and GbCML50 play a role in lignin accumulation.We detected significantly lower lignin spontaneous fluorescence under UV light and lighter colour of xylem using phloroglucinol-HCl staining in stem cross-sections in TRV:GbCML45 and TRV:GbCML50 plants when compared with TRV:00 plants after V. dahliae inoculation.
Additionally, the deposition of callose was also reduced in leaves of GbCML45-and GbCML50-silenced cotton plants after V. dahliae inoculation.These data indicated lower lignin and callose accumulation in TRV:GbCML45 and TRV:GbCML50 plants than in TRV:00 plants in response to Verticillium infection (Figure 7d).Consistently, the expression of several key genes involved in the lignin biosynthesis pathway, including GbPAL5, GbC4H1, GbC3H1, GbF5H2, GbCCoAOMT1 and GbCAD9, were significantly down-regulated in the TRV:GbCML45 and TRV:GbCML50 plants when compared with the corresponding value in TRV:00 plants (Figure 7e).The callose pathway genes β-1,3-glucanase genes 42 and 43 (GbGLU42 and GbGLU43), which are responsible for callose degradation and involved in Verticillium wilt resistance (Xu et al., 2016), were also significantly down-regulated in the TRV:GbCML50 and TRV:GbCML45 plants (Figure 7f).These results indicated that GbCML45 and GbCML50 may positively regulate lignin and callose syntheses to enhance cotton Verticillium wilt resistance through improving physical barrier defence.

| DISCUSS ION
Cytosolic Ca 2+ signalling plays a critical role in various aspects of plant growth, development and stress responses (Perochon et al., 2011).Perception of the Ca 2+ signal through the binding of Ca 2+ by the EF-hand Ca 2+ -motif of divergent forms of CaM/CML proteins can activate downstream components, triggering signal transduction (McCormack et al., 2005;Zhu et al., 2015).The expression patterns of CMLs vary according to plant developmental stages, tissue types and environmental stimuli, indicating their specific roles in plant growth, development and responses to abiotic and biotic stresses (Cheval et al., 2013;Moore et al., 2011;Ranty et al., 2016).In this study, we found that GbCML45 and its interacting partner GbCML50 are important positive regulators of plant resistance to V. dahliae infection.This finding was supported by the fact that knockdown of either GbCML45 or GbCML50 increased cotton susceptibility to V. dahliae infection (Figures 1 and   3).Further analyses elucidated that GbCML45 and GbCML50 are both localized in the cytoplasm and nucleus and act in a Ca 2+ -dependent manner to increase Verticillium wilt resistance (Figures 4 and 5).Earlier reports also showed that some CMLs, such as GhCML11 and GhCaM7 that are grouped in the same group III in the phylogenetic tree (Figure S5), function as positive regulators of upland cotton resistance to Verticillium wilt in a Ca 2+ -dependent manner (Cheng et al., 2016;Zhang et al., 2023).
However, GbCML45 and GbCML50 are located separately in groups IV and I, respectively, which are different from that of GhCML11 and GhCaM7, in the phylogenetic tree (Figure S5).
GhCML11 interacts with GhMYB108 TF and both act as positive regulators in cotton defence against V. dahliae infection (Cheng et al., 2016).In this study, we found that GbCML45 interacted with GbAP2-ERF and GbRAP2.3TFs in a Y2H assay, suggesting that different CMLs may have different interacting partners.Although silencing of GbAP2-ERF and GbRAP2.3caused an increase in disease symptom appearance on TRV:GbAP2-ERF and TRV:GbRAP2 plants (Figure 3j), the silenced plants did not show statistically significant differences, albeit an increased tendency, in disease index value when compared with TRV:00 plants (Figure 3i,j).No obvious differences in phenotype of brown spots and the biomass of V. dahliae in stems, as compared with those in TRV:00 plants, were found either (Figure 3k,l).These results suggest that the GbCML45-interacting GbAP2-ERF and GbRAP2 might have no or only a minor role in cotton resistance to V. dahliae infection.Taken together, CML proteins in cotton might interact and modulate the activities of some specific TFs, which in turn regulate the expression of downstream genes involved in cotton resistance to V. dahliae infection.
Biotic stresses have been reported to induce numerous cellular signalling events, including Ca 2+ flux, transcriptional regulation, and the biosynthesis and signal transduction pathways of defence-related hormones, such as SA, with the participation of CaMs/CMLs/CBPs (Zeng et al., 2023).In Arabidopsis, the knockout of pathogen-inducible CaM-binding protein CBP60g displayed an enhanced susceptibility to the bacterial pathogen P. syringae and reduced pathogen-induced SA accumulation, demonstrating a positive regulatory role of CBP60g in SA production and pathogen resistance (Wang et al., 2009).On the other hand, constitutive insect herbivore resistance and elevated levels of SA have been observed in the Arabidopsis loss-of-function mutant defective in signal responsive 1 (AtSR1), which encodes the calmodulin-binding transcription activator 3 (CAMTA3) (Aldon et al., 2018;Galon et al., 2008;Qiu et al., 2012;Rahman et al., 2016).This CaM-binding TF represses the expression of enhanced disease susceptibility 1 (EDS1), a positive component of SA signalling (Du et al., 2009;Qiu et al., 2012).Additionally, CAMTA3 and its binding CaM negatively regulate SA accumulation and SA signalling to increase Arabidopsis susceptibility to P. syringae and the fungal pathogen Botrytis cinerea (Galon et al., 2008;Rahman et al., 2016).In our study, we observed that the expression of GbCML45 and GbCML50 was induced by exogenous SA (Figure 6a), indicating that SA acts upstream of GbCML45 and GbCML50 as a positive regulator to trigger their expression.This finding further suggests that SA may potentially be involved in GbCML45/GbCML50-mediated cotton resistance to V. | 11 of 17 dahliae infection as a positive regulator.This premise was supported by the results that showed that the expression of NPR1 and PR genes, involved in SA signalling-related defence, were down-regulated in both GbCML45-silenced and GbCML50-silenced plants (Figure 6b).
Results of this study also indicated that the expression of GbCML45 and GbCML50 was induced by ET treatment (Figure 6a), suggesting that ET may also act upstream of GbCML45/GbCML50mediated cotton resistance to V. dahliae infection as a positive regulatory player similar to SA.This hypothesis was supported by two facts: (i) several signalling-related (e.g., GbEIN2 and GbERF1) genes were down-regulated in both GbCML45-silenced and GbCML50silenced cotton plants (Figure 6c) and (ii) several earlier studies also reported the positive role of ET signalling in cotton plant resistance to Verticillium wilt (Jia et al., 2022;Xiong et al., 2020;Yang et al., 2015).In addition, the ET biosynthesis-related genes such as GbACS6 and GbACO1 were down-regulated in GbCML45-silenced and GbCML50-silenced cotton plants (Figure 6c).These findings together suggest that GbCML45 and GbCML50 also regulate the expression of both the downstream genes of the ET signalling as well as the ET biosynthesis-related genes in a feedback manner to trigger plant defence in response to Verticillium wilt (Figure 8).
Roles of CaMs/CMLs/CBPs in ET-modulated plant defence against pathogen infections have also been observed in other plant species.For example, ET-mediated calcium-dependent protein kinase 2 (NtCDPK2) was shown to promote biotic stress in Nicotiana plants with the tomato resistance gene Cladosporium fulvum-9 (Cf-9) upon elicitation with the Avr9 peptide from the tomato pathogen C. fulvum, identified as an early gene-for-gene interaction race-specific elicitor (Ludwig et al., 2005).Another example showed that the CaM-binding TF AtSR1 enhanced plant defence against Pst DC3000 and ET-induced senescence by directly promoting the expression of ethylene insensitive 3 (EIN3) in Arabidopsis (Nie et al., 2012).
Both NO and ROS are important signalling molecules and act as downstream components in Ca 2+ signalling in plant immunity processes.Previous investigations revealed that NO acts downstream of the Ca 2+ signalling component in an early response to pathogen infection (Ali et al., 2007;Ma et al., 2008;Perochon et al., 2011).
There are strong lines of evidence showing that NO and ROS accumulation is induced by overexpression of Ca 2+ -CaM/CML proteins in the cytosol (Köster et al., 2022;Lamotte et al., 2004;Lecourieux et al., 2006;Ma et al., 2008;Ranty et al., 2016;Zeidler et al., 2004).For example, CaM1 promotes the production of NO, ROS and the expression of defence-related genes, leading to resistance to bacterial pathogen Xanthomonas campestris pv.vesicatoria in leaves of pepper (Choi et al., 2009;Kim et al., 2014).In cotton, the GhCaM7-enabled disease resistance to V. dahliae is related to the accumulation of NO and ROS (Zhang et al., 2023).This finding supports the positive correlation between reduced NO and ROS accumulation in GbCML45-silenced and GbCML50-silenced cotton plants and their susceptibility to V. dahliae infection (Figures 1d-i, 3a-f, and 7a-c).Collectively, GbCML45 and GbCML50 perceive the V. dahliae-induced increase in intracellular Ca 2+ , which leads to increased NO and ROS production through up-regulating the expression of related genes (Figure 7a-c).As a consequence, immunity responses are activated in cotton leading to Verticillium wilt resistance (Figure 8).
We found that lignin and callose accumulation was significantly reduced in stems of GbCML45-and GbCML50-silenced cotton plants after V. dahliae inoculation (Figure 7d), which might contribute to the weakened resistance of the silenced plants to V.
dahliae infection (Figure 7a), because the accumulation of lignin and callose is well known to contribute to enhanced resistance against V. dahliae in cotton through reinforcing cell wall structure (Huang et al., 2021;Mbiza et al., 2022;Xu et al., 2011).Similar results were also observed in other plant species in response to pathogen infection.For example, lignin deposition is rapidly induced after plant-pathogen interactions and enhances disease resistance to Pst DC3000 (AvrRpm1) in Arabidopsis (Kim et al., 2020;Lee et al., 2019).PAMPs (e.g., flagellin and chitin) induce cytosolic Ca 2+ influx and callose deposition in the elongation zone of primary roots, supporting a correlation between Ca 2+ level and callose accumulation (Dubiella et al., 2013).Arabidopsis AtCML9-overexpressing lines exhibit reduced susceptibility to P. syringae infection by deposition of callose and modification of callose biosynthesis-related gene expression (Leba et al., 2012).Consistently, the expression of AtCML41 was up-regulated by flagellin treatment, which facilitates callose deposition at plasmodesmata, and enhances plant defence against P. syringae (Xu et al., 2017).Our results suggested that lignin and callose deposition induced by GbCML45 and GbCML50 also plays a critical contribution to Verticillium wilt resistance in cotton.
The fast and efficient cotton transformation method using shoot apical meristem cell-mediated transformation system (SAMT; Ge

| Phylogenetic analysis and conserved domain architecture construction
MUSCLE in MEGA 7 was used to align GbCML45, GbCML50 and other related protein sequences (Kumar et al., 2016).To construct an unrooted phylogenetic tree, the neighbour-joining method and the Jones-Taylor-Thornton model were used through 1000 bootstrap replicates.To visualize the phylogenetic tree, Evolview (https:// www.evolg enius.info) was used (Balakrishnan et al., 2019).

| Cultivation of V. dahliae, gene cloning, VIGS vector construction and western blot analysis
The V. dahliae strain V991 was used in our experiment, which was highly aggressive, defoliating and stored in 30% glycerol at −80°C.
The activation and infection processes were same as previously described (Gao et al., 2013).To clone GbCML45 and GbCML50 genes, the respective coding sequences were obtained from the cotton database by sequence BLAST (https:// cotto nfgd.org/ seque ncese rver/ ).Primers were designed using Premier 6.0 (http:// www.premi erbio soft.com/ ) to amplify the GbCML45 and GbCML50 sequences from Hai7124.The detailed processes of plasmid construction, gene transformation and expression and detection of OE-GbCML45-GFP protein were described previously (Yi et al., 2023).

| Section anatomy in stems and fungal quantification
To observe sections of cotton plants, stems were cut into crosssections manually and a stereomicroscope (Olympus) was used for observation and photographing.Quantification of V. dahliae biomass was performed in accordance with previously described methods (Jia et al., 2022).

| RNA isolation, RT-qPCR and analyses and observation of subcellular localization
Total RNA was extracted from cotton leaves, stems and roots using the FastPure Universal Plant Total RNA isolation kit (Nanjing Vazyme Biotech Co., Ltd).The first-strand cDNA, qPCR assay, and relative gene expression level procedures were the same as previously described (Rieu & Powers, 2009;Yi et al., 2023) with the cotton UBQ7 gene (GhUB7, accession: DQ116441) used as the internal control.

| Y2H and BiFC assays
The yeast library construction and Y2H assays were conducted as

| Transcriptional activation assay
The coding sequences of GbCML45 and GbCML50, GbAP2-ERF and

| Cytosolic Ca 2+ signal detection
Cytosolic Ca 2+ staining and observation was performed as described by a previous study (Cheng et al., 2016).The fluorescence intensity of root cells was determined using ImageJ software.

| Hormone application
Chemicals of SA, ethrel (donor of ET) and MeJA were dissolved in ethanol to make 10 mM stock solutions.To prepare the working solutions, distilled water was added to dilute stock solutions to 10 μM, and same amount of ethanol was also added in distilled water as the control (mock).Three or four true leaves cotton seedlings were used for SA, ET and MeJA application by foliar spraying or soil drenching.Leaf and root samples were harvested and put into storage bags and frozen in liquid nitrogen and stored at −80°C for further analysis.Three biological replicates were used for each treatment.

| Histochemical assay and H 2 O 2 and NO measurement
Content of H 2 O 2 was examined by staining with DAB, which was determined using the method previously described (Wang et al., 2019).
H 2 O 2 and NO contents were determined by using detection kits (Solarbio), detailed operation steps were performed as in the manual instructions.All analyses had three biological replicates.

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Verticillium dahliae infection induces the expression of GbCML45 in roots of cotton, and silencing of GbCML45 reduces resistance to V. dahliae in cotton.(a) and (b) The fragments per kilobase of transcript per million mapped reads (FPKM) values of Gbscaffold26027.2.0 (GbCML45) in roots of resistant cultivar CSSL1 and susceptible cultivar CSSL4 of cotton.(a) Root-resistant cultivar mock (RRM) and root-resistant cultivar inoculated (RRI).(b) Root-susceptible cultivar mock (RSM) and root-susceptible cultivar inoculated (RSI).(c) The transcript levels of GbCML45 in the roots of island cotton (Hai7124) after 0, 0.25, 0.5, 1, 3, 5, 9 and 12 days post-inoculation (dpi) with V. dahliae infection.(d) The relative transcript levels of GbCML45 in roots of TRV:GbCML45 cotton plants.(e) Albino phenotype of the cotton plants inoculated with TRV:GbCLA vector after 2 weeks.(f, g) Disease symptoms and disease index of TRV:00 and TRV:GbCML45 seedlings at 21 dpi with V. dahliae.(h) Stem anatomy of TRV:00 and TRV:GbCML45 cotton plants shown in (f).(i) The relative biomass of V. dahliae in stems of TRV:00 and TRV:GbCML45 cotton plants.Data are means ± SD of three biological replicates (n = 3).Asterisks indicate statistically significant differences between TRV:00 and TRV:GbCML45 plants as determined by a Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001).
, implying that the roles of GbCML45 and GbCML50 in cotton plants might also be in charge of Ca 2+ -mediated stress signal transduction for cotton Verticillium wilt resistance.To test this hypothesis, we performed a time course V. dahliae infection experiment to assess the change of Ca 2+ levels in cotton root cytoplasm of TRV:GbCML45 F I G U R E 3 Silencing GbCML45-interacting protein genes reduced resistance to Verticillium dahliae in cotton.(a) Relative transcript levels of GbCML50 in roots of TRV:GbCML50 cotton plants.(b) The albino phenotype of the cotton plants inoculated with TRV:GbCLA vector.(c, d) Disease symptoms and disease index of TRV:00 and TRV:GbCML50 seedlings at 21 days post-inoculation (dpi) with V. dahliae.(e) Stem anatomy of TRV:00 and TRV:GbCML50 cotton plants shown in (c).(f) The relative biomass of V. dahliae in TRV:00, TRV:GbCML50 cotton plants.(g, h) Relative transcript levels of GbAP2-ERF and GbRAP2.3 in roots of TRV:GbAP2-ERF and TRV:GbRAP2.3 cotton plants, respectively.(i, j) Disease symptoms and disease index of TRV:00, TRV:GbAP2-ERF and TRV:GbRAP2.3 seedlings at 21 dpi with V. dahliae.(k) Stem anatomy and V. dahliae recovery culture assay.(l) Relative biomass of V. dahliae in stems of TRV:00, TRV:GbAP2-ERF and TRV:GbRAP2.3 cotton plants.Data are means ± SD of three biological replicates (n = 3).Asterisks indicate statistically significant differences between TRV:00 and TRV:GbCML50 plants as determined by a Student's t test (ns, p > 0.05, **p < 0.01, ***p < 0.001).and TRV:GbCML50 cotton plants.The Ca 2+ fluorescence of cotton root cells was measured through Fluo-4/AM indicator at 0, 5, 15, 30 and 60 min after the inoculation with V. dahliae.Our results showed that the fluorescence intensity in the root cells of TRV:00 control plants significantly increased to the peak at 5 min after V. dahliae inoculation, and then decreased quickly (Figure 5a), indicating that V. dahliae infection of cotton roots induced Ca 2+ influx into the cytosol.However, the fluorescence intensities in the cytosol of the root cells of TRV:GbCML45 and TRV:GbCML50 cotton plants were obviously weaker than in TRV:00 plants at the 5 and 15 min time points (Figure 5a, b), suggesting that the V. dahliae-induced Ca 2+ influx into the cytosol was impaired by the silencing of either GbCML45 or GbCML50.Consistently, the expression levels of several calcium signalling-related genes, including the calmodulin-binding IQ-domain protein (GbIQD1) and (GbIQD31), PINOID-binding protein (GbPBP1), CBL-interacting protein kinase (GbCIPK6) and Ca 2+ -binding protein EPS15 homology domain protein-encoding GbEHD2, were downregulated in both TRV:GbCML45 and TRV:GbCML50 plants compared with TRV:00 plants (Figure 5c-g).
sion levels of GbCML45 and GbCML50 following treatment with SA, ET and MeJA in cotton plants.Results revealed that the transcript levels of GbCML45 and GbCML50 were significantly increased by SA and ET treatments but decreased by MeJA treatment in cotton roots (Figure 6a), suggesting that the CML45-and CML50-mediated cotton Verticillium wilt resistance might depend on the SA and ET signals rather than the JA signal.To further explore the role of GbCML45/50 in SA and ET signalling pathways, we examined the expression of several defence-related marker genes involved in these pathways in roots of TRV:GbCML45, TRV:GbCML50 and TRV:00 cotton plants.These marker genes included GbNPR1 and GbPR1/5, involved in the SA signalling pathway, and GbACS6, GbACO1, GbEIN2 and GbERF1, involved in the ET signalling pathway.The transcript levels of all these investigated marker genes were significantly down-regulated in TRV:GbCML45 and TRV:GbCML50 cotton plants compared with the corresponding value in TRV:00 control plants (Figure 6b,c).These findings collectively suggest that GbCML45 and GbCML50 might mediate cotton Verticillium wilt resistance downstream of the SA and ET signals and activate the expression of defence-related genes involved in the SA and ET pathways.

Figure
Figure7ashowed that the ROS levels were lower in the leaves of the TRV:GbCML45 and TRV:GbCML50 plants than in those of TRV:00 plants at 24 and 48 h post-inoculation (hpi) with V. dahliae.The hydrogen peroxide (H 2 O 2 ) contents were also significantly lower in the roots of TRV:GbCML45 and TRV:GbCML50 plants than in those of TRV:00 plants, with the maximum decrease being observed at 24 and 48 hpi (Figure7b).In addition to the reduced ROS accumulation,

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I G U R E 6 Relative transcript levels of GbCML45 and GbCML50 after treatment with hormones salicylic acid (SA), ethylene (ET) and methyl jasmonate (JA), and hormones or defence-related genes in roots of TRV:00, TRV:GbCML45 and TRV:GbCML50 Hai7124 plants.(a) Relative transcript levels of GbCML45 and GbCML50 in roots of Hai7124 plants after treatment with 5 μM SA, ET or JA for 0, 2, 4, 8, 12 and 24 h.(b, c) Relative transcript levels of several defence-related genes in SA and ET signal pathways, respectively, in roots of TRV:00, TRV:GbCML45 and TRV:GbCML50 Hai7124 plants.The expression value in TRV:00 plants was normalized as 1.Data are means ± SD of three biological replicates (n = 3).Asterisks indicate statistically significant differences between TRV:00, TRV:GbCML45 and TRV:GbCML50 plants as determined by a Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001).

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I G U R E 7 Accumulation of reactive oxygen species (ROS), lignin, callose and their production-related genes expression in GbCML45-and GbCML50-silenced Hai7124 cotton plants after inoculation with Verticillium dahliae.(a) 3,3′-diaminobenzidine (DAB) staining of GbCML45and GbCML50-silenced cotton leaves to detect the O 2 •− accumulation at 24 and 48 hours post-inoculation (hpi) with V. dahliae.(b) Content of H 2 O 2 in the GbCML45-and GbCML50-silenced cotton roots at 24 and 48 hpi with V. dahliae.(c) Relative transcript levels of ROS productionrelated genes and NO content in the GbCML45-and GbCML50-silenced cotton roots at 48 hpi with V. dahliae.(d) Lignin spontaneous fluorescence under UV light and xylem phloroglucinol-HCl staining in stem cross-sections, and aniline blue staining of callose in leaf of GbCML45-and GbCML50-silenced cotton plants.(e, f) Relative transcript levels of lignin and callose biosynthesis-related genes in GbCML45and GbCML50-silenced cotton roots.Data are means ± SD of three biological replicates (n = 3).Asterisks indicate statistically significant differences between TRV:00, TRV:GbCML45 and TRV:GbCML50 plants as determined by a Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001).

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I G U R E 8 A proposed model for the role of GbCML45 and GbCML50 in regulating cotton resistance to Verticillium dahliae via interaction with salicylic acid (SA)-and ethylene (ET)-signalling pathways and promoting reactive oxygen species (ROS) accumulation and lignin biosynthesis.V. dahliae infection induces the influx of Ca 2+ , which combines with the heterodimer GbCML45 and GbCML50 or homodimer GbCML45 and GbCML45, resulting in increasing ROS accumulation and NO production, and subsequently transmitting the defence signals into nucleus; simultaneously the signals are enhanced by SA and ET.In the nucleus, the expression of GbAP2-ERF, GbAP2.3,NPR1 and ERF1 enhances expression of PR genes and lignin biosynthetic genes, resulting in increased cotton V. dahliae resistance.Black arrows indicate stimulation, black blunt bars indicate suppression, dotted arrows indicate uncertain stimulation.
et al., 2023) could be used to create cotton materials overexpressing GbCML45 and GbCML50 to enhance the disease resistance of cotton varieties.In conclusion, our results illustrated that V. dahliae infection induces the influx of Ca 2+ into cytosol, which activates GbCML45 and GbCML50 and the related TFs, resulting in increased ROS and NO accumulation, up-regulated expression of genes involved in SA and ET signalling pathways like NPR1 and ERF1, up-regulated expression of PR genes and accumulation of lignin and callose, leading to enhanced cotton V. dahliae resistance (Figure8).Our results provide new insights into the Ca 2+ -CML signalling-mediated cotton Verticillium wilt resistance, with the example of GbCML45 and GbCML50, which eventually will lead to new targets for molecular breeding cultivars with improved resistance to Verticillium wilt in cotton.

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Plant materials and growth conditionsIn this study, the island cotton cultivar Hai7124 and the upland cotton cultivar Jimian11 were used.For germination, seeds of cotton were first imbibed in water for 8 h at 28°C in the dark in an incubator.Three days later, the germinating seedlings were transferred to soil in pots under an 8 h light 22°C/16 h dark 25°C photoperiod/temperature cycle for 10 days.These 10-day-old seedlings were then used for VIGS infiltration.For Arabidopsis, the Columbia-0 (Col-0) ecotype was used as the WT, and the ectopically expressing GbCML45 or GbCML50 Arabidopsis transgenic lines were obtained using the vector of pCAMBIA2300-GFP.Seeds of Col-0 background Atcml46 (SALK_127471C) and Atcml49 (SALK_035905C) were obtained from Salk Institute Genomic Analysis Laboratory (http:// signal.salk.edu/ ).

GbRAP2. 3
were fused with the pGBKT7 vector to generate BD-GbCML45, BD-CML50, BD-AP2-ERF and BD-RAP2.3.The plasmid pGBKT7-53 was used as positive control.Each vector was transformed into the Y2H gold yeast strain and plated on SD/−Trp (SDO) medium for positive selection.Dilutions of yeast clones were then plated onto SD/−Trp/−His/−Ade (TDO) and TDO/X-Gal media for subsequent transcriptional activation assays.Images were taken after incubation on the medium for 3 days.